Kuntai Capsules Improve Premature Ovarian Failure by Regulating AMPK-Mediated Autophagy

Abstract

Background: Premature ovarian failure (POF) is a gynecological endocrine disorder with current treatments having limitations. Kuntai capsule (KTC), a traditional Chinese medicine formulation, is thought to be beneficial for POF, but its mechanism is unclear. Network pharmacology can help explore drug mechanisms.

Methods: A POF rat model was established using cyclophosphamide (CTX). Rats received low-dose KTC (0.6 g/kg/d), high-dose KTC (1.8 g/kg/d), or dehydroepiandrosterone (DHEA, positive control). Ovarian function was evaluated via histopathology, hormone assays (ELISA), apoptosis (TUNEL/flow cytometry), autophagy markers (Western blot), and network pharmacology.

Results: KTC treatment (especially high-dose) ameliorated POF in CTX-treated rats, as shown by increased ovarian weight, restored estrus cycle, and improved follicle development. The serum estradiol (E2), anti-mullerian hormone (AMH), and superoxide dismutase (SOD) levels increased, whereas the follicle-stimulating hormone (FSH), malondialdehyde (MDA), and reactive oxygen species (ROS) levels decreased following KTC treatment. KTC also alleviated ovarian cell apoptosis and autophagy, with higher-dose KTC being more effective. Network pharmacology predicted AMPK/mTOR pathway involvement. Western blot confirmed KTC activated the AMPK/mTOR signaling, downregulated autophagy markers (LC3B-II/I, Beclin1), and upregulated P62. Autophagy inhibition (via 3-MA) mirrored KTC effects, while mTOR blockade (rapamycin) reversed them.

Conclusions: KTC ameliorates POF by inhibiting excessive ovarian autophagy through AMPK/mTOR pathway activation, providing a mechanistic basis for its clinical use.

Keywords: AMPK/mTOR signaling pathway; Autophagy; Follicular granulosa cell; Kuntai capsule; Premature ovarian failure.

Fig. 1

Effects of Kuntai capsules on the body weights and ovaries of rats. A: Curves of body weight changes in different groups of rats within 28 days; B: Ovary weights of the rats; C: Rat ovarian indices; D: Regular curves and average length histograms of the estrus cycles of rats of the rats in different groups; E: H&E staining of ovary sections from the different groups of rats. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Effects of Kuntai capsules on follicle and blood vessel formation in rats. A: The number of follicles in the rats; B: Western blot results of rat ovarian growth-related proteins; C: Immunohistochemical images of the rat ovarian tissue sections. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Effects of Kuntai capsules on hormone levels and aging in rat ovarian tissue. A: ELISA analysis of the serum hormone, oxidative metabolite and oxidase levels in the rats; B: Immunohistochemical map of the expression of markers of ovarian aging in the rats. *P < 0.05, **P < 0.01

Fig. 4

Effects of Kuntai capsules on ovarian apoptosis and autophagy in rats. A: Ovarian cell apoptosis was analyzed by flow cytometry and TUNEL staining. B: Relative expression of rat ovarian aging marker proteins. C: Relative expression of apoptosis marker proteins in rat ovarian tissue. D: Relative expression of autophagy marker proteins in rat ovarian tissue. *P < 0.05, **P < 0.01

Fig. 5

Network pharmacological enrichment analysis of Kuntai capsules

Fig. 6

Kuntai capsules affect ovarian autophagy through the AMPK-mTOR pathway. A: Ovarian cell apoptosis was analyzed by flow cytometry and TUNEL staining. B: Relative expression of apoptosis marker proteins in rat ovarian tissue; C: Relative expression of autophagy marker proteins in rat ovarian tissue; D: Relative expression of AMPK‒mTOR pathway marker proteins in rat ovarian tissue. *P < 0.05, **P < 0.01, ***P < 0.001, ^ns P > 0.05